Anti-freeze proteins (AFPs) have been suggested for improving the freezing tolerance of foodstuffs.
For the purpose of the invention, the term AFP has the meaning as well-known in the art, namely those proteins which exhibit the activity of inhibit the growth of ice crystals. See for example U.S. Pat. No. 5,118,792.
WO 90/13571 discloses antifreeze peptides produced chemically or by recombinant DNA techniques. The AFPs can suitably be used in food-products. Example 3B shows modified ice crystal shapes if a water-ice mixture is frozen into a film in combination with 0.01 wt % of AFP.
WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal shape in ice-cream. This document also describes a process for extracting a polypeptide composition from extracellular spaces of plants by infiltrating leaves with an extraction medium without rupturing the plants.
WO 94/03617 discloses the production of AFPs from yeast and their possible use in ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
Several literature places also mention the isolation and/or use of plant proteins for cryoprotection. Cryoprotective proteins have a function in the protection of plant membranes against frost damage. These proteins, however, do not possess recrystallisation inhibition properties and are, therefore, not embraced within the terms AFPs.
Hincha in Journal of Plant Physiology, 1992, 140, 236-240 describes the isolation of cryoprotective proteins from cabbage.
Volger in Biochimica et Biiophysica Acta, 412 (1975), 335-349 describes the isolation of cryoprotective leaf proteins from spinach.
Boothe in Plant Physiol (1995), 108: 759-803 describes the isolation of proteins from Brassica napus. Again, these proteins are believed to be cryoprotective proteins rather than AFPs.
Neven in Plant Molecular Biology 21: 291-305, 1993 describes the DNA characterisation of a spinach cryoprotective protein.
Salzman in Abstracts and Reviews of the 18th Annual Meeting of the ASEV/Eastern Section in Am. J. Enol. Vitic., Vol. 44, No. 4, 1993 describes the presence of boiling-stable polypeptides in buds of Vitis. Although the proteins are analogous to fish antifreeze peptides, they are cryoprotective proteins and not AFPs.
Lin in Biochemical and Biophysical Research Communication, Vol. 183, No. 3, 1992, pages 1103-1108 and in Lin, Plant Physiology (1992) 99, 519-525 describes the 15 kDa cryoprotective polypeptide from Arabidopsis Hakaira.
Houde in The Plant Journal (1995) 8(4), 583-593 mentions cryoprotective proteins from wheat.
Furthermore--as illustrated in example VIII--extracts of cabbage, spinach, Brassica napus and Arabidopsis do not have recrystallisation inhibition proteins after heating.
Up till now, however the use of AFPs has not been applied to commercially available food products. One reason for this are the high costs and complicated process for obtaining AFPs. Another reason is that the AFPs which until now have been suggested for use in frozen food products cannot be incorporated in the standard formulation mix, because they tend to destabilise during processing especially during the pasteurisation step. This destabilisation is believed to be caused by the denaturation of the AFPs; this is a well-known effect commonly observed for peptides and proteins.
The present invention aims at providing solutions to these problems.
Surprisingly it has been found that AFPs can be isolated from natural sources such as cold-acclimatised plants by means of a new relatively simple process. This process leads for the first time to the identification of AFPs which can conveniently be incorporated in a mix for the preparation of frozen products before the pasteurisation thereof.
Accordingly in a first aspect, the invention relates to a process for the recovery of AFPs from natural sources, said process involving the steps of
a) isolating a AFP containing juice from the natural source; PA1 b) heat treating the natural source or the AFP containing juice to a temperature of at least 60.degree. C.; PA1 c) removing the insoluble fraction.
Step c of the above process will usually take place after steps a and b. Step a and b can be done in any desired order, for example step a followed by step b (in that case the AFP rich juice will be heated) or step b followed by step a (in that case the natural source will be heated) or step a and b simultaneously.
Surprisingly we have found that the isolation process of the invention has a number of advantages.
Firstly by using the process it is no longer necessary to avoid rupturing of the natural source such as plants such as required in the processes according to WO 92/22581. This immediately significantly increases the commercial applicability of the process, for example as compared to WO 92/22581, because high investment costs for specific processing are no longer necessary.
Also by using the high temperatures it seems possible to extract from a large group of peptides present in the natural sources a new selection of very active AFPs from the natural material, said AFPs including peptides which are very active w.r.t. ice-recrystallisation inhibition properties.
Thirdly, contrary to expectations, the use of high temperatures does not denature all the proteinaceous material, but does only seem to denature some of the proteins, while the remaining AFPs have an increased temperature stability. This renders it possible to include the isolated AFPs in compositions which need to be subjected to higher temperatures e.g. a pasteurisation step. This is especially surprising, because for example the AFPs from WO 92/22581 appear not stable under heating conditions (see example VI).
The process of the invention includes in step b the heating of the natural source or the AFP rich juice to a temperature of more than 60.degree. C. Preferably the temperature is from 60 to 110.degree. C., most preferably from 80 to 105.degree. C. The heating step can take place after the isolation of the protein rich juice (step a) or before the isolation of the protein rich juice. Any suitable way to heat the juice can be used, for example conventional or microwave heating, heating optionally with an added extraction medium, steaming etc.
If an extraction medium is used, preferably it is used in small volumes to avoid unnecessary dilution of the AFP fraction. Any suitable extraction medium can be used, although the use of water is especially preferred. If desired, additives may be added to the water prior to using it as an extraction medium. Most preferred, however water substantially free of additives is used.
The process of the invention can be applied to any natural source of heat-stable AFPs. Included in this list are plants, fishes, insects and microorganisms. Both natural occurring species may be used or species which have been obtained through genetic modification. For example microorganisms or plants may be genetically modified to express AFPs and the AFPs may then be isolated in accordance to the present invention. AFPs having at least 80%, more preferred more than 95%, most preferred 100% homology to the AFPs directly obtained from natural sources can thus be obtained. For the purpose of the invention proteins possessing this high level of homology are also embraced within the term AFPs. Also these transformed microorganism or plants capable of expressing genes encoding the AFPs are also embraced within the scope of the invention.
Genetic manipulation techniques may be used to produce the heat stable AFPs described in the invention. An appropriate host cell or organism would be transformed by a gene construct that encodes the desired heat stable polypeptide. The nucleotide sequence coding for the heat stable polypeptide can be inserted into a suitable expression vector containing the necessary elements for transcription and translation and in a manner that they will be expressed under appropriate conditions (eg in proper orientation and correct reading frame and with appropriate targeting and expression sequences). The methods required to construct these expression vectors are well known to those skilled in the art.
A number of expression systems may be utilised to express the heat stable polypeptide coding sequence. These include, but are not limited to, bacteria, yeast insect cell systems, plant cell culture systems and plants all transformed with the appropriate expression vectors.
A wide variety of plants and plant cell systems can be transformed with the nucleic acid constructs of the polypeptides isolated in the heat stable extract. Preferred embodiments would include, but are not limited to, maize, tomato, tobacco, carrots, strawberries, rape seed and sugar beet.
Preferably the AFP is derived from plants (this means that either the AFP is directly obtained from the plant as natural source or AFPs having a high degree of homology to these AFPs are transgenetically produced in other organisms). Any plant containing heat stable AFPs can be used, preferably however are naturally occurring plants (or their genetic modified versions) which are able to grow under cold conditions such that they contain AFPs. Especially preferred is the use of winter-rye, perennial grasses and sedges. Other suitable plants may for example come from the group of woody plants, winter-cereals etc.
Especially preferably the heat stable AFPs are derived from Acer saccharoides, Bamboo, Buddleia, Isothecium myosuroides, Ramalina farinaceae, Usnea subfloridana, Forsythia, Oxalis, Poa Trivialis, Lolium Perenne, Holcus Lanatus, Bromus Sterilis, Parodiochloa flabellata, Deschampsia antartica, Carex aquatilis, Colobanthus quintensis and Agrostis tenuis, Festuca contracta and Poa annua.
The AFP rich juice can be separated from its source by any convenient process for example pressing, filtering, homogenising, extraction etc. Preferably the natural source of AFP such as the plant material is made into small pieces or into a slurry before the protein rich fraction is collected, for example by filtering. This maceration can be done by any suitable method, for example in a blender. It will be well within the ability of the skilled person to divide the material into such a form that collection of the protein rich juice can readily take place.
After collecting and heating (in the desired order) the protein fraction the resulting AFP containing sample can then be treated by any convenient process in order to remove the insoluble fraction and retain the AFP rich liquid fraction. The insoluble fraction can be removed e.g. by filtering, precipitation etc. The AFP rich liquid can then advantageously be further processed to concentrate or isolate the AFPs to bring them in a form suitable for further use. Examples of suitable processes are drying to obtain a powder or paste, further concentration to obtain an AFP concentrate, chromatography to separate the AFPs from the extraction medium etc. Again it will be well within the ability of the skilled person to determine the suitable means and conditions for appropriate isolation.
For some natural sources the AFPs obtained by the above methods may consist of a mixture of two or more different AFPs. If desired these AFPs can be separated by any conventional process for example chromatography or other processes based on the differences in physical/chemical properties such as molecular weight.
Also if desired the amino acid composition and sequence of the isolated AFPs can be determined. Any suitable method for determining these can be used. Examples of suitable methods are described in the examples. Also if desired the nucleic acid sequence that encodes the AFPs can be determined. Vector containing a nucleic acid sequence capable of encoding the amino acids are also embraced within the scope of the invention.
Based on the above information it is also possible to genetically modify other natural sources such that they produce the advantageous AFPs as identified hereabove. Examples of suitable AFPs are described in the examples.
It has been found that the AFPs obtained by the above process have an increased ability to withstand thermal treatment. It is believed that such AFPs have never before been isolated. As described above this increased thermal resistance is particularly of interest for use in food-products which undergo a heating step, for example pasteurisation.
Accordingly another aspect of the invention relates to AFPs which have a thermal stability as evidenced by no significant reduction in the recrystallisation inhibition properties after heat-treatment for one hour at 80.degree. C. or 10 minutes at 100.degree. C. A suitable test for determining the ice recrystallisation inhibition properties is described in the examples and involves the quick freezing to -40.degree. C. followed by storage for one hour at -6.degree. C. Preferably AFPs which are subject to this test after heat-treatment result in an ice crystal particle size which is less than 5 .mu.m larger than the ice crystal size of a sample with the same AFP which was not heat-treated. Preferably the difference is less than 3 .mu.m, most preferred less than 1 .mu.m.
Preferably those AFPs are chosen which have significant ice-recrystallisation inhibition properties. A suitable test for determining the recrystallisation inhibition properties is indicated in the example VI. Preferably AFPs in accordance to the invention provide a ice particle size following an ice recrystallisation inhibition assay--as described in the examples--of 15 .mu.M or less, more preferred from 5 to 15 .mu.m.
The AFPs can conveniently be used in food products, preferably in food products which are frozen or intended to be frozen. Especially preferred is the use of AFPs in products which are heated e.g. by pasteurisation or sterilisation prior to freezing. Especially preferred is the use in frozen confectionery products.
Examples of such food products are: frozen confectionery mixes such as ice-cream mixes and water-ice mixes which are intended to be pasteurised prior to freezing. Such mixes are usually stored at ambient temperature. Suitable product forms are for example: a powder mix which is packed for example in a bag or in sachets. Said mix being capable of forming the basis of the frozen food product e.g. after addition of water and optionally other ingredients and--optional--aeration.
Another example of a suitable mix could be a liquid mix (optionally aerated) which, if necessary after addition of further components and optional further aeration can be frozen.
The clear advantage of the above mentioned mixes is that the presence of the AFP ingredient makes that the mixes can be frozen under quiescent conditions, for example in a shop or home freezer without the formation of unacceptable ice crystal shapes and hence with a texture different to products normally obtained via quiescent freezing.
Very conveniently these mixes are packed in closed containers (e.g. cartons, bags, boxes, plastic containers etc). For single portions the pack size will generally be from 10 to 1000 g. For multiple portions pack sizes of up to 500 kg may be suitable. Generally the pack size will be from 10 g to 5000 g.
As indicated above the preferred products wherein the AFPs are used are frozen confectionery product such as ice-cream or water-ice. Preferably the level of AFPs is from 0.0001 to 0.5 wt % based on the final product. If dry-mixes or concentrates are used, the concentration may be higher in order to ensure that the level in the final frozen product is within the above ranges.
Surprisingly it has been found that compositions of the invention can contain very low amounts of AFPs while still being of good quality.
Surprisingly it has been found that the level of AFPs can be as low as 0.1 to 50 ppm while still providing adequate recrystallisation properties and temperature tolerance in frozen confectionery products. Although applicants do by no means wish to be bound by any theory, the reason for this may be that the interaction between the solids of the frozen confectionery and the AFPs provides an excellent mechanism for inhibiting crystal growth. Most conveniently the level of AFP is from 1 to 40 ppm, especially preferred from 2 to 10 ppm.
For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as ice-cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees. For some applications the use in fermented food products is less preferred.
Preferably a the level of solids in the frozen confection (e.g. sugar, fat, flavouring etc) is more than 4 wt %, for example more than 30 wt %, more preferred from 40 to 70 wt %.
Frozen confectionery products according to the invention can be produced by any method suitable for the production of frozen confectionery. Especially preferably however all the ingredients of the formulation are fully mixed before pasteurisation and before the freezing process starts. The freezing process may advantageously involve a hardening step, for example to a temperature of -30 Fahrenheit or lower.